Method and device for high field strength electrotransfection of microvescicles and cells

ABSTRACT

A device, system and process involve conducting electroporation of microvesicles or exosomes or other target structures in a microfluidic arrangement at pressures that exceed atmospheric pressure. Single as well as multiple flow configurations can be employed. In some cases, the system and its operation are computer-controlled for partial or complete automation.

RELATED APPLICATIONS

This application claims the benefit under 35 USC 119(e) of U.S.Provisional Application No. 62/726,691, filed on Sep. 4, 2018, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Many applications in biology, medicine, pharmaceutical research andother areas use techniques in which genetic materials are introducedinto cells. The term “transformation” is often used when working withbacteria or non-animal eukaryotic cells, including plant cells.“Transduction” requires virus-mediated gene transfer into eukaryoticcells, while “transfection” refers to gene delivery by means other thana virus (chemical transfer, electrotransfer, etc).

Biological materials of interest include not only DNA, siRNA, mRNA, RNPcomplexes, but also small molecules or proteins such as antibodies. Inmany cases, the process involves opening transient pores or “holes” inthe cell membrane to allow the uptake of the “cargo” material and thusalter or genetically modify the cells or cell products.

One common technique used to temporarily permeabilize cells, typicallyby applying an electric pulse, is electroporation. Parameters consideredwhen developing electroporation procedures include cell properties (cellsize, shape, membrane structure, surface charge, for example), the cellenvironment, and attributes of the applied electric field, (e.g., pulseintensity, number of pulses, pulse duration, pulse shape and/orfrequency). It is generally believed that membrane permeabilizationduring electroporation occurs after the applied electric field induces athreshold value in the transmembrane potential or “electroporationthreshold” and that, at a given applied electric field, there is athreshold for the number of pulses and pulse length, needed forsuccessful electroporation. The Schwan equation and related derivationsare often used to estimate a cell's transmembrane potential thatdevelops in response to relevant experimental parameters includingapplied field, cell size, conductivities of media, cellular cytosol, andcell membrane, and membrane thickness (“Analytical Description ofTransmembrane Voltage Induced by Electric Fields on Spheroidal Cells”,Biophysical Journal Volume 79 August 2000 670-679).

Attractive for potential uses in prognosis, therapy, or as biomarkers,exosomes are cell-derived vesicles that are present in many if not alleukaryotic fluids, including blood, urine, as well as cell culturemedia. Generally, exosomes are composed of lipid bilayer membranes withmultiple adhesive proteins on their surface. Known for theircell-to-cell communication characteristics, it is thought that exosomesmay find applications in targeted cell therapy.

Whereas eukaryotic cells typically have a diameter within the range offrom about 10 to about 100 microns (μm), typical microvesicles andexosome diameters are between 30 and 1000 nanometers (nm). At suchreduced dimensions, exosomes are also smaller than most prokaryoticcells (0.1-5.0 μm in diameter).

Various electroporation devices have been developed and are commerciallyavailable. However, they are generally designed and/or optimized forprokaryotic and/or eukaryotic cells.

It is generally believed that existing electroporation systems cannotachieve the field strengths thought to be required for exosomeelectroporation; approximately 100-300 kV/cm predicted by the Schwanequation. Although some academic papers (see, e.g., Nucleic AcidsResearch, 2012, Vol. 40, No. 17 e130, or “Delivery of siRNA to the mousebrain by systemic injection of targeted exosomes”, Nature Biotechnology,20 Mar. 2011, doi:10.1038/nbt.1807) appear to indicate loading ofexosomes at low field strengths (˜1-10 kV/cm), it is uncertain ifluminal loading was actually achieved in these studies. It ishypothesized that luminal loading would protect the therapeutic agentfrom in vivo degradation mechanisms, and allow for a higher precisiondelivery method.

SUMMARY OF THE INVENTION

Attempting to apply existing electroporation approaches to exosomesraises various difficulties. One major impediment relates to physicaldifferences in the relevant length scales for electroporation, withcellular diameters spanning a range several orders of magnitude largerthan exosomes. Assuming the Schwan equation (“Analytical Description ofTransmembrane Voltage Induced by Electric Fields on Spheroidal Cells”,Biophysical Journal Volume 79 August 2000 670-679) is a valid model oftransmembrane potential, exosome equivalent transmembrane potentials arenot accessible with commercially available electroporation systems.Specifically, high field strengths of 100-300 kV/cm are required toachieve transmembrane potentials in the 0.2 to 1 Volt range and variablepulse lengths (10 nanoseconds-1,000 microseconds) may be targeted.

In addition, commercially available electroporation devices exposebiologics to direct contact with electrodes, resulting in potentialdamage due to local heating and Faradaic by-products (hydronium ions,hydroxyl ions, chlorine, free radicals, and electrode breakdownby-products (e.g. aluminum ions and particulate)). Existingelectroporation approaches lack microfluidics to transport heat awayfrom thermally susceptible biological entities. They also lackco-localization of exosomes and payload, leading to inefficient use ofpayload. Absent too is a high throughput of transfection.

A need exists, therefore, for equipment and procedures that targetand/or facilitate the electroporation of exosomes. A need also existsfor approaches that address one or more of the deficiencies discussedabove.

Generally, the invention relates to transferring (uploading orunloading) materials into or out of target structures such as, forinstance, exosomes, other vesicles or even cells. In specific aspects,permeabilization of the target structures is conducted byelectroporation techniques in a system that includes a stream containingthe target structures under pressures exceeding atmospheric pressuresand generally at field strengths above the dielectric breakdown strengthof the same fluid under atmospheric pressures.

Flow patterns can be supported by microchannels and, in someembodiments, the invention relates to a device designed to direct flowssuch as those described above through an electric field. Additionally,or alternatively, the device provides an individual inlet andcorresponding outlet for each flow. In one example, separateinlets/outlets are provided for directing the central flow, a firstinner sheath flow, a second inner sheath flow, a first outer sheath flowand a second outer sheath flow.

Practicing the invention facilitates the electroporation of exosomes andaddresses problems encountered with conventional systems. Robust,flexible and versatile, embodiments of the invention can be applied oradapted to materials other than exosomes, cells or other vesicles, forinstance. Principles described herein also can be employed to removesome or all of the contents of target structures; that is, opening poresand allowing the internal contents to diffuse out either passively orvia active electrophoretic forces.

In general, according to one aspect, the invention features anelectroporation system. The system comprises an electroporation devicecomprising a flow channel and a pumping system for generating controlledpressures and flow rates through the flow channel undergoingelectroporation. The pressure are elevated, exceeding two atmospheres.

In some cases, the pressures are higher than 100 pounds per square inchor 690 kPa, or higher than 1000 pounds per square inch or 6900 kPa, orhigher than 2000 pounds per square inch or 13800 kPa.

In a current example, the pumping system comprises a syringe pump. AHPLC (High Pressure Liquid Chromatography) pump could also be used.

A receiver device is used to control back pressure in the flow channel.

In a current example, the electroporation device comprises a top block,a bottom block, and a dielectric spacer separating the top block and thebottom block. Typically, the blocks have a thickness that is greaterthan 5 millimeters to provide adequate rigidity. The spacer preferablycomprises a flow channel cutout in the spacer for defining lateral sidesof the flow channel. A profile of the flow channel can be narrow at eachend with a wider center.

In general, according to another aspect, the invention features anelectroporation device comprising a top block, a bottom block, and adielectric spacer separating the top block and the bottom block andhaving a flow channel cutout for defining lateral sides of a flowchannel.

In general, according to another aspect, the invention features anelectroporation method comprising providing a flow channel, generatingpressures of greater than two atmospheres in the flow channel, andelectroporating targets, such as microvesicles or exosomes, in the flowchannel.

In general, according to another aspect, the invention features anelectroporation system comprising a receiving device providing abackpressure in a flow channel, an input device for pumping a fluidcontaining targets into the flow channel at a desired flow rate, and anelectroporation device for electroporating targets in the flow channel.

In general, according to another aspect, the invention features anelectroporation method comprising providing a backpressure at a flowchannel, pumping a fluid containing targets into the flow channel at adesired flow rate, and an electroporation of targets in the flowchannel.

The above and other features of the invention including various noveldetails of construction and combinations of parts, and other advantages,will now be more particularly described with reference to theaccompanying drawings and pointed out in the claims. It will beunderstood that the particular method and device embodying the inventionare shown by way of illustration and not as a limitation of theinvention. The principles and features of this invention may be employedin various and numerous embodiments without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, reference characters refer to the sameparts throughout the different views. The drawings are not necessarilyto scale; emphasis has instead been placed upon illustrating theprinciples of the invention. Of the drawings:

FIGS. 1A and 1B are a perspective exploded view and a perspective viewof an electroporation device for a single flow according to the presentinvention;

FIG. 2A is an image showing an example of a prototype pumping systemused to achieve high pressures with controlled flow rates duringelectroporation;

FIGS. 2B and 2C are plots of electric field and current as a function oftime showing the maximum electric field and example current traces,respectively, at pressures of 14.7 and 2,000 pounds per square inch(psi).

FIG. 2D is an additional image showing the example of the prototypepumping system of FIG. 2A;

FIG. 3 is a schematic diagram of a system employing a device and pumpingsystem that can be used to achieve high pressures with controlled flowrates during electroporation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention now will be described more fully hereinafter withreference to the accompanying drawings, in which illustrativeembodiments of the invention are shown. This invention may, however, beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will be thorough and complete, and willfully convey the scope of the invention to those skilled in the art.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items. Further, the singular formsand the articles “a”, “an” and “the” are intended to include the pluralforms as well, unless expressly stated otherwise. It will be furtherunderstood that the terms: includes, comprises, including and/orcomprising, when used in this specification, specify the presence ofstated features, integers, steps, operations, elements, and/orcomponents, but do not preclude the presence or addition of one or moreother features, integers, steps, operations, elements, components,and/or groups thereof. Further, it will be understood that when anelement, including component or subsystem, is referred to and/or shownas being connected or coupled to another element, it can be directlyconnected or coupled to the other element or intervening elements may bepresent.

It will be understood that although terms such as “first” and “second”are used herein to describe various elements, these elements should notbe limited by these terms. These terms are only used to distinguish oneelement from another element. Thus, an element discussed below could betermed a second element, and similarly, a second element may be termed afirst element without departing from the teachings of the presentinvention.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

The invention generally relates to approaches for transferring one ormore material(s), referred to herein as “cargo” or “payload”, into orout of target structures (also referred to herein as “targets”).Specific aspects relate to microfluidic techniques suitable for theelectroporation of vesicles such as microvesicles and exosomes, forinstance. Other target structures that can be used include various celltypes as well as other materials that can be loaded with cargo viaelectroporation techniques.

The target structures can be characterized by their size. Some have adiameter that is less than or equal to about 100 nm, for instance withinthe range of from about 30 to about 1000 nm. Other suitable targets havea diameter that is within the range of from about 10 to about 100 μm. Infurther cases, the target structures have a diameter within the range offrom about 0.1 to about 5.0 μm.

In many embodiments the target is nanoparticles, i.e., vesicles having adiameter equal or less than 100 nanometers (nm), such as, for example,within the range of from about 30 to about 100 nm. In other embodiments,the target is a microparticle, e.g., a cell, having a diameter greaterthan about 100 nm. In many cases, the microparticle can have a diameterof up to 25, 50, 75, or 100 μm. T-cells, a common target forelectroporation transfection, for example, range in size from about 6 μmto 12 μm.

The targets can be provided in a suitable buffer, as known in the art oras developed for a particular application. This buffer can be referredto as the targets' “preferred” buffer. Examples include but are notlimited to commercially available buffers such as BTX CytoporationT-media and those known in the literature such as “Low-ConductivityBuffers for High-Sensitivity NMR Measurements”, J. AM. CHEM. SOC. 2002,124, 12013-12019, “The influence of medium conductivity onelectropermeabilization and survival of cells in vitro”,Bioelectrochemistry 54 Z̆2001. 107-115.

The cargo is a suitable material that can be incorporated into thetargets. Examples include but are not limited to small molecules,chromosomes, DNA, RNA, RNP's e.g., mRNA, siRNA, gRNA, ssRNA, CrisperCas9), other genetic materials, oligomers, biomarkers, proteins,transposons, biomolecule complexes, small molecules, therapeutic agents,and so forth. In one illustrative example siRNA is loaded into exosomesas a cancer therapy to knock down oncogenes in vivo.

Cargo to be introduced in the targets can be provided in one or moreelectroporation buffers or solutions. Many cargo-containing buffers arecommercially available. In some cases, a suitable solution can beprepared using, for example, techniques and ingredients known in theart.

In further implementations, principles described herein are applied oradapted to removing some or all of the contents from the targets,vesicles, for instance. In this situation, opening the vesicle poresallows the internal contents to diffuse out either passively or viaactive electrophoretic forces. Releasing contents from the targets maybe particularly useful with cargo that is insufficiently characterized,thus raising potential regulatory or other concerns during therapeuticdevelopment.

In many of its aspects, the invention relates to permeabilizing targetstructures such as microvesicles, cells, exosomes or other membranebound structures in a device or system in which the target structuresare present in a fluid flow or stream at a pressure greater than theatmospheric pressure. In general, microvesicles are a type ofextracellular vesicle that are released from the cell membrane andreside in the interstitial space between cells and in many types of bodyfluids including blood. Microvesicles range in size from 30 nanometers(nm) in diameter to 1000 nm in diameter. Microvesicles are generallylarger, on average, than exosomes, which range in size from about 30 nmto about 200 nm.

A device for a single flow configuration (in which target structures canbe provided in a suitable buffer) is illustrated in FIGS. 1A (explodedview) and 1B. In this configuration, two blocks form the top wall andthe bottom wall of a flow channel and electroporation electrodes,respectively.

In more detail, the electroporation device (also referred to herein as“electroporation chamber”) 3 includes top plate or block 13 and bottomplate or block 15, which can be formed of conductive metals that arebiocompatible and bio-inert or conductive metals that are coated with abiocompatible bio-inert metal. Examples of biocompatible bio-inertmetals include platinum, stainless steel (316 stainless steel, forexample), nitinol (a metal alloy of nickel and titanium), andcobalt-chrome. Examples of structural materials that could be coatedwith biocompatible bio-inert coatings include, copper and aluminum.Other materials such as rigid plastics (Ultem, PEEK, COC), ceramics,glass, or silicon that have a conductive metal coating may also be usedto construct block 13 and 15. In some cases, it may be advantageous tomake the structural block from a transparent material to visualize theflow.

Usually, each of the blocks must be rigid to withstand the generatedpressures. As a result, in many embodiments, each of the blocks is metaland has a thickness T that is greater than 5 millimeters.

Two O-ring grooves 17A, 17B are formed in the bottom block 15 and seateither a single or concentric O-rings OR to enable high fluidic pressuresealed operation. A single O-ring can be employed in some embodiments.

In the illustrate example, six (6) holes 50-1 to 50-6 surround theO-ring grooves 17A, 17B. These holes 50-1 to 50-6 receive respectiveclamping bolts. The holes 50-1 to 50-6 of the top block 13 are sized toaccept respective hollow cylindrical bolt insulators 51-1 to 51-6 thatelectrically isolate the clamping bolts from the bottom block 15.

In the center of the O-ring grooves 17A, 17B is a planar upper (fluidchannel) face 52 of the lower bottom block 15.

A polymer sheet (also referred to herein as “polymer spacer”) 19 islaser cut to define the block separation via its thickness and toprovide cutouts 54-1 to 54-6 for the clamping bolts, bottom blockelectrode cutout 55, an alignment pin cutout 63, and the fluidic flowchannel cutout 60.

In general, selection of the specifications and the patterning of hispolymer sheet 19 allows rapid prototyping of the fluid channel's keyparameters including the channel geometry. For example, the channelgeometry includes length, width, height, and profile. These areimportant both for creating a uniform flow through the channels (e.g. nodead zones) and also for controlling the electrical requirements toachieve a given field (i.e. the current and voltage), repetition rate(relating to residence time and other factors). Additional parametersare electrode separation, fluid residence time (in conjunction with theflow rate), the system resistance to match the impedance range of thepower supply, and the surface area to volume (important for boundarylayer effects and heat transfer. In the illustrate example, the profileof the channel is narrow at each end with a wider center.

In general, one key feature to be considered when selecting and/ordesigning polymer sheet 19 concerns the need to form an adequate seal inconjunction with the O-rings used in O-ring grooves 17A, 17B. Anotherkey feature relates to providing a higher dielectric strength relativeto that of the fluid, to prevent arcing.

Various materials can be employed to make polymer sheet 19. Examplesinclude but are not limited to elastomers such as polyurethane, siliconeor Paralyene (which is the trade name for a variety of chemical vapordeposited poly polymers used as moisture and dielectric barriers). Insome cases, the Paralyene formulation selected has a relatively highdielectric strength of greater than 1000 Volts/millimeter and preferablyabout 7,000 Volts/millimeter.

The polymer sheet may be patterned in areas outside of the O-ringlocations (see grove 17B in FIG. 1A) or may form one seating surface ofthe O-ring seal on the top block. Alternatively, a polymer coating maybe patterned directly on the electrode blocks to form the flow channel.

The top block 13 includes a mirror arrangement of O-ring grooves to theO-ring grooves 17A, 17B of the bottom block 15 to receive its O-rings ORthat engage the top side of the sheet 19. The top block also includes acorresponding array of holes 56-1 to 56-6 that mirror the pattern of theholes 50-1 to 50-6 of the bottom block. The holes 56-1 to 56-6 of thetop block 13 are sized to accept respective hollow cylindrical boltinsulators 57-1 to 57-6 that electrically isolate the clamping boltsfrom the top block 13.

Suitable bolts 70-1 to 70-6, see FIG. 2A, extend through the hollowcylindrical bolt insulators 51-1 to 51-6 and bolt insulators 57-1 to57-6 to clamp together the blocks 13 and 15. Other arrangements, asknown in the art, can be used to hold together these blocks whileoperating at a pressure higher than atmospheric.

The flow channel 21 is laterally defined by the fluidic flow channelcutout 60 formed in the sheet 19. When fully assembled, the bottom ofthe flow channel is defined by the upper center face of the bottomblock, and the top of the flow channel is defined by the lower centerface of the top block 13.

Fluid enters the flow channel 21 (a microfluidic channel, for example)through inlet 23 provided by a tube connector 58 that is inserted intothrough-hole 59 which has been threaded in the top block 13. Thethrough-hole terminates at and opens into the proximal end of the flowchannel 21.

Fluid then exits the flow channel 21 through outlet 25 provided by atube connector 60 that is inserted into a second threaded through-hole61 in the top block 13 that terminates at and opens into the distal endof the flow channel 21.

The top block 13 and the bottom block 15 are controlled electrically viaa top block electrode 31 and a bottom block electrode 33. The top blockelectrode 31 is pressfit into a blind hole 61 in the top block 13. Thebottom block electrode is surrounded by a hollow cylindrical electrodeinsulator 64 that isolates it from the top block 13. It extends throughthe top block 13 and through the electrode cutout 55 in the polymersheet 19. The bottom block electrode 33 makes electrical contact withthe bottom block 15 by being press fit into a blind hole 65 formed inthe bottom block 15.

In operation, the fluid is exposed to one or more than onepermeabilizing electric pulses and then flows out of the device 3through the outlet 25. Blocks 13 and 15 are charged from a voltagefunction generator that functions as an electroporation power supply 75via the electrodes 31 and 33.

FIG. 2A is photograph of a prototype pumping system used to achieve highpressures during electroporation. Referring to FIG. 2A, a programmablesyringe pump [1] controls the flow rate from a high pressure stainlesssteel syringe [2] into the inlet of the electroporation chamber [3]. Theoutlet of the electroporation chamber is connected to a receivingstainless steel syringe pump [4]. A fixed back pressure is provided bythe receiver syringe through the force applied to the syringe plunger bya pneumatic actuator [5] connected to a pneumatic regulator.

This simple system allows both a controlled pressure and a controlledflow rate, both of which are required to allow precise electrical fieldexposure to the fluid without arcing. A BTX Gemini electroporation powersupply and function generator 75 is connected to the electroporationchamber [3] to provide a sequence of electrical pulses at fixedintervals. A custom firmware was developed for the Gemini allowing anunlimited number of pulses to be delivered at programmable pulse ratefrom 0.1 to 100 seconds, pulse widths of 10-1,000 microseconds, andvoltages from 300-3,000 volts. A computer controller 76 with a 12-bitNiDAQ digital to analog converter card 77 measures the voltage acrossthe electrodes and the current through the electrodes using an analoginterface 78 that included a series 0.1 Ohm sense resistor allowing thecurrent waveform for each electroporation pulse to be captured foranalysis. A three-way ball valve (not shown) can be added to the outletto allow system sampling filling, rinsing, and purging. The entirewetted path can be sterilized with standard techniques includingautoclave, ETO, or gamma irradiation.

FIGS. 2B and 2C show the maximum electric field and example currenttraces respectively at 14.7 and 2,000 psi. The conditions for all testsinclude a 50 microsecond pulse duration at approximately 1 Hz using theexosome solution with a conductivity of 0.02 S/m. The buffer in FIG. 2Bhas a conductivity of 0.01 S/m. The maximum field strength achievable isinversely related to the solution conductivity; higher conductivitieswill arc at lower field strengths and deionized water will arc at asignificantly higher field strength.

FIG. 2D presents a different view of the system in FIG. 2A or sectionsthereof.

While this system enables proof of concept studies, one skilled in theart could implement a number of alternatives or improvements includinghaving all systems under computer control and synchronization (pumpingsystem, back pressure adjustment, valve control, current monitoring, andelectrical pulse control). In situ sensors, such as, for example, fluidpressure sensors, fluid flow sensors, and electrical current sensors todetect pressure, flow and current in the channels and between theelectrodes, are further preferably included. Sensors can enable closedloop control over the system.

Other high pressure pumping systems could be used including HPLC (highperformance liquid chromatography) pumps with flow rate and pressuresensors for either the inlet and/or the outlet. Alternatively, the inletand outlet pressure and flow rates could be controlled with highpressure regulators and pressure and flow rate sensors to enable closedloop control.

In general, in the preferred embodiment, the pressure in the flowchannel 21 exceeds two atmospheres. Often the pressure is much higher,however, such as higher than 100 psi or 690 kPascals (kPa), and even ashigh as 1000 psi or 6900 kPA to 2000 psi or 13800 kPa or to as high as4000 psi or 27600 kPa, or more. Currently 2000 psi is being used.Theoretically, for the dielectric breakdown of water, the higher thepressure the better.

There should be upper limits on the pressure, nevertheless. The upperlimit may be dictated by the biology. When exposed to high pressure,many proteins, for example, will denature and/or deactivate. Generally,this occurs at >100 MPa (14,500 psi). Accordingly, the pressure in theflow channels will typically be less than 14,500 psi.

While not shown, a sheath flow can keep the biological entities awayfrom boundary layer/wall effects, resulting in a more uniform residencetime in the area undergoing electroporation.

The sheath flow may be planar by for example adding two additionalinputs and outputs or an axisymmetric or sheath flows in the vertical aswell has horizontal planes can be utilized, resulting in improvedresidence time uniformity and thus the anticipated transfectionuniformity. Axisymmetric and non-axisymmetric arrangements can beimplemented in the flow channel or via suitably designed tubing from thepumps. For example, a dual lumen coaxial catheter could be used tocreate an axisymmetric inner sheath flow surrounding the centerbiological flow. The sheath flows can have a similar conductivity as thecentral flow or a different conductivity. For instance, sheath flowswith a higher or lower conductivity may be used to direct the fieldlines optimally through the biologic containing central flow. Theoptimal sheath composition and geometry can be simulated using COMSOLFEA including fluid mechanics, diffusion, electrophoresis, andelectrical field simulations.

In a device such as device 3 in FIGS. 1A and 1B, sheath flowarrangements can be implemented by adding one or more fluidic inlet(s)and outlet(s), e.g., similar to fluidic inlet 21 and fluidic outlet 23.In some situations, pathways can be formed in the substrate (polymerspacer 19 in FIG. 1A) to connect a sheath inlet to the entrance sectionsof channel 21 and the sheath outlet to the exiting section of channel21.

Multiple flow arrangements are described, for example, in U.S. patentapplication Ser. No. 16/400,270, filed on May 1, 2019 entitled Methodand Device for Exosomes Electroporation, which is incorporated herein bythis reference in its entirety. One example includes: a central stream,inner sheath streams at either side of the central stream and outersheath streams at the exterior boundary of the inner sheath streams. Intypical arrangements, the center stream fluid (including targets) andthe inner sheath fluid (a first buffer, for instance) have a low σ,while the outer sheath fluid (e.g., a second buffer) has a high σ.Another example includes a center stream (target-containing fluid)having a low σ disposed between sheath streams (buffer) having a high a.

With multiple streams, mixing between streams can be prevented, reducedor minimized by maintaining flows in the laminar regime. While variousstreams are in physical contact with one another (for transferring cargoto the targets, for instance), flow patterns can be controlled orfacilitated by microchannel arrangements configured into the devicesupporting the flows.

The device and/or pumping system described above can be part of a systemsuch as shown in the schematic diagram of FIG. 3 . Shown in this figureis system 100, which includes a reservoir 101 for holding targetstructures in a suitable culture medium or buffer. In some cases, thereservoir is an incubator. An agitator can be provided to stir thetarget structures and prevent them from settling. Other fluids that maybe needed to conduct a specific process can be supplied from additionalreservoirs. The FIG. 3 embodiment shows two illustrative reservoirs,namely upstream reservoir 105 and downstream reservoir 107. More orfewer reservoirs can be employed, however. If needed, reservoirs such as105 and/or 107 can supply cargo, buffers, washing or rinsing fluids,purging fluids, and so forth. In some cases, the cargo can be providedtogether with the targets being supplied from fluid reservoir 101.

An input pump 1 (e.g., programmable syringe pump 1 as in FIG. 2A, a HPLCpump or another suitable pump) directs targets-containing fluid fromreservoir 101, which is the fluid volume of the syringe of the syringepump, to electroporation device 3. Typically, the targets aremicrovesicles or exosomes.

In specific implementations, the electroporation device 3 has the singleflow configuration shown in FIGS. 1A and 1B and the target containingfluid is directed to fluidic inlet 23. Electroporation is conductedusing electroporation power supply and function generator 75 whichproduces suitable pulses applied to blocks 13 and 15 via electrodes 31and 33.

Output fluid is drawn from electroporation device 3 by the receivingsyringe 4 or another suitable device. The pneumatic actuator 5 sets thebackpressure. In current examples, the backpressure is set to be higherthan 100 pounds per square inch or 690 kPa. In some examples, thebackpressure is higher than 1000 pounds per square inch or 6900 kPa.

The controller 76 sets and maintains the desired pressure in theelectroporation device 3 by controlling the receiving device to set theback pressure in the flow channel 21 of the electroporation device 3.

In general, the receiving device must have compliance to receive fluidat a constant pressure. Other examples include a pressure vesselpossibly containing an immiscible fluid to prevent gas from dissolvingin the electroporated fluid.

Then the controller 76 sets and maintains the desired flow rate bycontrolling the input pump to pump against the backpressure at thedesired flow rate.

With a fixed backpressure, the controller 76 then operates the pump 1 toprovide the desired flow rate while controlling the electroporationpower supply 75 to apply the desired voltages across the blocks 13, 15of the electroporation device 3.

In the single flow configuration of device 3 (FIGS. 1A and 1B) output isdrawn from fluidic outlet 25.

Pressure regulators or other approaches for maintaining a desired fluidpressure during electroporation can be employed.

If one or more additional reservoirs are present, each can be providedwith its own pump to supply other regents and buffers. In one example,reservoir 105 is provided with pump 115 and reservoir 107 is providedwith pump 117.

Output (including, for instance, cargo-loaded target structures, can becollected in reservoir 119, which can be an incubator. In an operationcontrolled by the controller 76, after the targets are received into thereceiving syringe 4, a valve 130 to the device is closed and thecontroller operates the pneumatic actuator 5 to slowly depressurize thereceiving syringe to ambient pressure, then the receiving syringe isactuated to eject the targets into the reservoir 119. If present, sheathfluids can be collected in waste reservoir 125 or directed to anarrangement for recycle. Reservoir 125 also can receive other spentfluids such as washing and/or rinsing solutions.

The system is controlled by controller 76. Often the controller is amicroprocessor in a computer system such as a single board computersystem. In other cases, the controller is a microcontroller withintegrated memory and analog to digital converters and digital to analogconverters. In some embodiments, the system is partially or completelyautomated, with the controller 76 controlling one or more of pumps,incubator conditions, flow patterns, electrical function generator 75,various valves (not shown), such as valves that open or close access toand from various reservoirs, sensors such as sensors 123 comprising oneor more probes or detectors for monitoring, setting, adjusting and/ormaintaining various parameters such as voltage magnitudes and pulseprofiles, temperatures, flow conditions, pressures, incubatorconditions, and so forth.

In specific implementations controller 76 receives measurements fromvarious sensors 123 and digitizes these measurements using the analog todigital converter 77 and uses these measurements to adjust processparameters such as flow attributes (rates, residence time, laminar vs.turbulent profile, alignment of the flow streams on their intended path,etc.) In one example, at least one of sensor 123 relies on electrodessuitably placed to allow measurements of the fluid properties (e.g.conductivity or field strength) to optimize the flow parameters in realtime of each of the streams individually. Further one or more of sensors123 can be employed to measure stream temperatures, field strengthsduring electroporation, pressures, and/or other process parameters ofeach of the streams, individually.

Operation of the system can be conducted by an optional initial washingor sterilization of device 3. The controller 76 opens valves orenergizes the pump 1 and other pumps if additional fluids are employed,to supply central and sheath streams, for example. Controller 76 alsoactivates the electrical function generator 75 which provides anelectrical potential across the electroporation electrodes and adjustsprocess parameters based on preset inputs or commands from the operatoror on information received from sensors 123. Product is collected atoutput reservoir 119 while spent sheath fluids is collected from device133 and directed to waste reservoir 125 or recycled.

Chosen residence times can vary from 100 microseconds (μs) to about asecond. An AC (for example, sinusoids or pulse trains with periods/pulsewidths ranging from 10 ns to 100 s of microseconds) or DC electric fieldis established and remains active while targets flow through the device.The magnitude of the field is tuned for the specific type of target to avalue sufficient to achieve permeabilization. In specific examples, thefield is in the range of 100-30,000 kV/m.

Suitable conductivity values for the central fluid can be within therange of from about 0.1 to about 0.01 S/m (Siemens per meter).

In addition, the low conductivity fluid minimizes Joule heating, animportant consideration for biologics. The temperature rise due to jouleheating is given by:

${\Delta\; T_{1}} = \frac{\sigma_{0}V_{0}^{2}t_{p}}{\rho\; c\; d^{2}}$where σ₀ is the conductivity, V₀ is the applied voltage, t_(p) is thepulse duration, ρ is the fluid density, c is the heat capacity and d isthe gap between the electrodes. Joule heating in this case is directlyproportional to the solution conductivity.

Use of microfluidic flow allows convective transport of heat generatedvia Joule heating.

Another characteristic of some of the microfluidic approaches describedherein involves electrodes that are remote from the biological entities.By keeping the electrodes far from the biological entities relative todiffusional length/time scales, potentially damaging Faradaicby-products (oxygen, hydronium, chlorine, free radicals) cannot interactwith the biological entities nor can the biological entities undergodirect redox reactions at the electrodes.

In general, higher field strengths are reached by minimizing thegeneration of arc nucleating vapor bubbles at the electrodes. This canbe accomplished by several strategies. In one embodiment, the fluid ismaintained under a high pressure (1-1,000 bar) which prevents bubblesfrom nucleating and collapses any existing air bubbles. Also minimizingthe current required to generate a given field and/or by spatiallycontrolling the field strengths can reduce arcing from air bubbles. Insome implementations, the current is minimized by using one or more lowconductivity fluids to simultaneously minimize gaseous electrolysisproducts (Faraday's law), while also minimizing Joule heating which canlead to local boiling. Additionally, by minimizing the field strength inthe vicinity of the electrodes, any bubbles that do form are less likelyto initiate an arc by exceeding the vapor dielectric breakdown voltage(˜30 kV/mm in air).

Further embodiments described herein relate to mitigating the formationof bubble, via electrolysis products, for example. Various techniquescan be employed. One approach relies on degassing the fluid e.g., in theregion where the electrodes reside, to dissolve or prevent nucleation ofgaseous electrolysis by-product.

Another approach relies on the electrode capacitance (e.g. higherelectrode surface area) thereby operating in a capacitive mode andminimizing or eliminating Faradaic current. In turn, electrode surfacearea can be increased by: increasing electrode nominal size; increasingelectrode effective electrochemical area by roughening the target;depositing nanoclusters in vapor phase; or by electrochemicallydepositing rough films (e.g. platinum black).

Current can also be capacitively coupled through a non-conductive sheathfluid. In this case, metal electrodes could charge a thin non-conductivesheath fluid which is in communication with the central flow. Forexample, ethylene glycol is a high dielectric constant non-conductivefluid. Alternatively, the non-conductive sheath flow could be immisciblewith the central flow such as an oil phase.

Other approaches for controlling bubbles use PEDOT (PSS orpoly(3,4-ethylenedi-oxythiophene) polystyrene sulfonate) or otherconducting polymer or metal (e.g., Ag) that undergoes Faradaic chargetransfer.

Sense electrodes can be placed within the outer, inner, or central flowsto make relevant measurements (e.g. conductivity, temperature, fieldstrength measurements). These electrodes may provide real time feedbackto adjust the operational parameters during electroporation. As anexample, an RTD electrode may be placed in the central flow to monitorthe temperature excursion of the biologics. A further example mayinclude placing electrodes to measure the conductivity of the sheath andcentral flows at the outlet to allow non-visual alignment of the flowsto the intended outlets and to make sure there is not excessive mixingof the flows. A further example may include placing opposing electrodesacross the central flow to measure the potential difference therebyestimating the field strength in the central flow.

Operationally, the pressure and fluid velocities can be nominallymatched at the stream interfaces; having separate outlets for each fluidallows the pressure drop to the outlet for each flow stream to beindependently tuned to prevent the fluid streams from deforming and/ordeflecting. Additionally, common inlets for sheath flows can beundesirable in terms of creating electrical leak paths by providingcompeting electrical paths with the intended electrical path across thecentral fluid.

In some cases, flows can be visualized to allow parameters to be tunedin real time using tracers or imaging the biological entities iftransparent electrode blocks are used (e.g. transparent plastic or glasswith patterned electrodes. Alternatively, electrodes can be placedlocally at the beginning and end of the flow channels to allowmeasurements of the fluid properties (e.g. conductivity or fieldstrength) to optimize the flow parameters in real time such that fluidpaths remain aligned to the intended flow paths. This tuning can bepassive by designing channel geometries or outlet tubing with theappropriate diameters and lengths or by actively applying pressuresusing a chamber with a pressure controller.

In some cases, target structures can be driven, e.g., acoustically, fromone buffer to another by performing buffer exchanges such as described,for example, in U.S. patent application Ser. No. 16/359,626, filed onMar. 20, 2019, entitled Acoustically-Driven Buffer Switching forMicroparticles, or U.S. patent application Ser. No. 16/557,820 filed onAug. 30, 2019, entitled Method and Apparatus for High Throughput HighEfficiency Transfection of Cells, both being incorporated herein byreference in their entirety.

Implementations of the invention can be practiced or adapted toreagent-based methods such as delivery by lipids (e.g. transfectamine),calcium phosphate precipitation, cationic polymers techniques,DEAE-dextran, magnetic beads, virus-based approaches, and otherapplications.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. An electroporation method comprising: providing aflow channel; and generating pressures of greater than two atmospheresof pressure in the flow channel; and electroporating targets in the flowchannel while the targets are under the greater than two atmospheres ofpressure by applying an electric field to the electroporation targets inthe flow channel using a voltage generator and electrodes associatedwith the flow channel.
 2. The electroporation method of claim 1, whereinthe pressures are higher than 100 pounds per square inch or 690 kPa. 3.The electroporation method of claim 1, wherein the pressures are higherthan 1000 pounds per square inch or 6900 kPa.
 4. The electroporationmethod of claim 1, wherein the pressures are higher than 2000 pounds persquare inch or 13800 kPa.
 5. The electroporation method of claim 1,further comprising providing back pressure in the flow channel.
 6. Theelectroporation method of claim 1, wherein the targets are microvesiclesor exosomes.
 7. The electroporation method of claim 1, furthercomprising after electroporating the targets, releasing a pressure ofthe targets to ambient pressure and providing the targets into areservoir.
 8. The electroporation method of claim 7, further comprisingsupplying a buffer into the reservoir to preserve the targets.
 9. Theelectroporation method of claim 1, wherein providing the flow channelcomprises providing an electroporation device defining the flow channel.10. The electroporation method of claim 9, wherein generating thepressures comprises operating a pumping system to generate controlledpressures and flow rates through the flow channel in the electroporationdevice undergoing electroporation.
 11. The electroporation method ofclaim 10, wherein the pumping system comprises a syringe pump.
 12. Theelectroporation method of claim 10, wherein the pumping system comprisesa receiver device for controlling back pressure in the flow channel. 13.The electroporation method of claim 9, wherein providing theelectroporation device comprises providing a top block, a bottom block,and a dielectric spacer separating the top block and the bottom block.14. The electroporation method of claim 13, wherein the blocks have athickness that is greater than 5 millimeters.
 15. The electroporationmethod of claim 13, wherein the dielectric spacer comprises a flowchannel cutout in the spacer for defining lateral sides of the flowchannel.
 16. The electroporation method of claim 15, wherein a profileof the flow channel cutout is narrow at each end with a wider center.17. The electroporation method of claim 1, wherein targets have adiameter that is within the range of 30 to 1000 nm.